Cancer immunotherapy is a versatile and rapidly expanding field as novel treatment approaches are constantly evaluated based on their ability to boost the host’s immune system to combat cancer cells. The adaptable drug affinity conjugate (ADAC) platform is an immunotherapy approach developed to customize personalized peptide-based therapeutic cancer vaccines. The ADAC technology utilizes an agonistic anti-CD40, bispecific, tetravalent antibody (BiY) conjugated to a peptide-antigen. However, data generated when studying a peptide-based therapeutic like the ADAC platform in vitro correlates poorly with the in vivo situation. Therefore, we aim to optimize an enzyme-linked immune absorbent spot (ELISpot) assay to better mimic the in vivo impact on peptide therapeutics and to better understand the ADAC technology. Briefly, human peripheral blood mononuclear cells (PBMC) were conditioned with a well-characterized CD8 epitope with or without BiY in an ELISpot assay to quantify antigen-specific T-cells activation. Assay optimization was conducted by examining adjustments to the protocol, such as decreasing the temperature, and exposure time to stimuli, as well as including a protease or a pre-stimulation period prior to the ELISpot assay. Although assay optimization was shown to be a time-consuming process, the inclusion of a pre-stimulation period of PBMCs using the ADAC technology showed to enhance T-cell activation upon re-stimulation. Moreover, it was shown that the peptide-Tag (pTag), enabling linkage of the antigen cargo and the antibody, as well as BiY, in the absence of the CD8 epitope, are not sufficient to generate a T-cell recall response. Furthermore, in addition to illustrating the complexity of assay optimization, this project contributes to a more comprehensive understanding of the ADAC platform.